5'-terminal structure of poliovirus polyribosomal RNA is pUp.

Hewlett, Martinez J.; Rose, John K.; Baltimore, David

doi:10.1073/pnas.73.2.327

Cited by 223 publications

(94 citation statements)

References 18 publications

(9 reference statements)

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“…Our results, therefore, indicate that the presence of an inverted nucleotide at the 5' end of mRNA is not required for translation in eucaryotic systems. This conclusion is in agreement with the recent finding of Baltimore and associated [16] that polio virus mRNA, isolated from polysomes of infected cells, lacks 7-methylguanosine.…”

Section: Discussionsupporting

confidence: 82%

Translation of Enzymically Decapped Messenger RNA

Abraham

Pihl

1977

European Journal of Biochemistry

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An enzymic procedure was used to remove the 7-methylguanosine diphosphate moiety at the 5' ends of ratbit hemoglobin mRNA and mouse immunoglobulin light-chain mRNA. Evidence was obtained that the procedure, which involves the use of polynucleotide kinase, does not result in any further degradation of the mRNA.The enzymically decapped mRNA was as effective as untreated mRNA in supporting protein synthesis in a wheat germ system. This was the case over a wide range of mRNA concentrations and over a considerable period of time. The presence in the incubation mixture of S-adenosylhomocysteine, an inhibitor of methylation, did not affect the results.The data indicate that the presence of a 7-methylguanosine diphosphate residue at the 5' end of mRNAs is not an obligatory requirement for translation in eucaryotic systems.It has recently been discovered [1,2] that many eucaryotic mRNAs and heterogeneous nuclear RNAs (hnRNAs) have at the 5' end of the chain certain characteristic features, so-called capped structures. These consist of an 'inverted' 7-methylguanosine, attached to the rest of the messenger through a 5'-5'-triphosphate linkage. Moreover, one or two of the subsequent nucleotides may be methylated in the C-2 positions of the ribose residues.Attempts have been made to elucidate the biological significance of the capped structures by studying the template activity of messengers where the 7-methylguanosine residue has been removed by chemical methods. Thus, Shatkin and coworkers [3] treated rabbit globin messenger and vesicular stomatitis virus mRNA with periodate/aniline, which specifically removes ribose moieties having free hydroxyl groups at the 2 and 3 positions, and hence removes the inverted nucleoside and the 3'-terminal nucleoside. Since the ability of the treated messengers to serve as templates in a protein-synthesizing system was strongly reduced, they concluded that the capped structure is required for normal functioning of the mRNA. It should be realized, however, that periodate/aniline treatment of a capped mRNA leaves abnormal structures at the termini of the chain, viz. a triphosphate group at the 5' end and a phosphate groups at the 3' end of the molecule.Recently we have developed an enzymic procedure which specifically splits the pyrophosphate Abbreviations. m'GDP, 7-methylguanosine diphosphate; Hepes, 4-(2-hydroxyethyl)-l -.piperazineethanesulphonic acid.linkage between m7GDP and the rest of the mRNA [4]. Here we have applied this method to rabbit globin mRNA and mouse immunoglobin light-chain mRNA, and demonstrated that the decapped mRNAs retain their full ability to serve as templates in a protein-synthesizing system. MATERIALS AND METHODS ChemicalsPhosphorylcreatine and creatine kinase were purchased from Calbiochem. Dithiothreitol, ATP, GTP and non-radioactive amino acids were obtained from Sigma Chemical Co. Radioactive amino acids were obtained from the Radiochemical Centre (Amersham, England). 7-Methylguanosine diphosphate was a gift from Dr A. J. Shatkin. Preparation of Crude Immunog...

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Section: Discussionsupporting

confidence: 82%

Translation of Enzymically Decapped Messenger RNA

Abraham

Pihl

1977

European Journal of Biochemistry

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“…A portion (25 ,ul) of each extract was analyzed by electrophoresis through a 15% SDS-polyacrylamide-0.09% N,N'-methylenebisacrylamide gel. Lanes 1,3,5,8,11,14, and 16, mock-infected extracts (-); lanes 6, 9, 12, 15, and 17, mutant-infected extracts (M); lanes 2, 4, 7, 10, and 13, wild-type-infected extracts (WT). (B) Portions (25 pul) of CV-1 extracts shown in panel A were resolved on a 15% SDS-polyacrylamide-0.09% N,N'-methylenebisacrylamide gel, and the resolved proteins were then transferred to nitrocellulose.…”

Section: Resultsmentioning

confidence: 99%

“…We used a cDNA copy of poliovirus type I to create a mutation in the protein 8 (Fig. 5B) (27) which contains the active site serine.…”

Section: Discussionmentioning

confidence: 99%

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Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

Bernstein¹,

Sonenberg²,

Baltimore³

1985

Mol. Cell. Biol.

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169

141

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A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.It has long been recognized that infection of cells with poliovirus results in a selective inhibition of host cell protein synthesis (for a review, see reference 2). Translation of cellular mRNA steadily declines as virus-specific translation increases; by 2 to 3 h after infection viral RNA, which unlike most other translated eucaryotic RNAs lacks a 5' m7Gp-ppN(m) cap structure (8,28), is translated almost exclusively. Presumably, a viral product mediates this inhibition, but it has not been identified.Results of early studies have shown that translation of cellular mRNA is blocked at the initiation step (19). More specifically, the defective initiation factor appears to be a salt-stable complex composed of a cap-binding protein (CBP) and associated proteins. This complex is thought to be crucial for the attachment of capped mRNAs to 40S ribosomal subunits (for a review, see reference 37). Infected cell extracts do not support the translation of capped RNAs (7,34) or their attachment to the CBP complex (16), but both activities can be restored by the addition of CBP complex from uninfected cells (17, 41). In vivo, the decline in host protein synthesis is preceded by specific cleavage of a 220,000-dalton protein (P220) which is part of the CBP complex (6). It has been proposed that the complex is inactivated by this cleavage (6). A viral product is thought to carry out or promote the cleavage of P220, but the known viral protease (protein 7c) is pr...

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“…Picornaviruses, however, do not possess cap structures (Hewlett et al, 1976;Lee et al, 1976) and in those for which sequence data are available the initiating AUG is not the 5' proximal one. Indeed, Forss et al (1984) have shown for the 0, serotype of FMDV that eight additional AUGs are located between the poly(C) tract and the initiation sites.…”

Section: B E C L a R K E A N D Othersmentioning

confidence: 99%

Two Initiation Sites for Foot-and-Mouth Disease Virus Polyprotein in vivo

Clarke¹,

Sangar²,

Burroughs

et al. 1985

Journal of General Virology

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SUMMARYTypically, the translation of eukaryotic mRNAs into protein is initiated at a single site. However, we have recently shown that not one but two primary products, P20a and P I6, are translated from the 5' end of the coding region of the genome of foot-andmouth disease virus (FMDV). In this paper we show by partial protease digestion of these proteins that they differ only at their N termini, thus confirming the presence of two initiation sites for translation of FMDV RNA. Sequence analysis of two subtypes of the virus (Al0 and A12) confirms the presence of two initiator AUG codons in the expected position on the genome. By correlation with protein synthesis data from these subtypes it appears that the relative use of each initiation site is dependent on its surrounding nucleotide sequence. In addition, the ratio of the two proteins when synthesized in vitro differs markedly from that when they are synthesized in vivo, suggesting the presence of a control mechanism for synthesis of P20a in vivo which may be absent in vitro. We also show that the cleavage site between these two proteins and the structural protein precursor, P88, is located closer to the N terminus of the polyprotein than has previously been reported.

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5'-terminal structure of poliovirus polyribosomal RNA is pUp.

Cited by 223 publications

References 18 publications

Translation of Enzymically Decapped Messenger RNA

Translation of Enzymically Decapped Messenger RNA

Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

Two Initiation Sites for Foot-and-Mouth Disease Virus Polyprotein in vivo

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